Protocol for mapping tumor infiltration by murine T cells using multiplex immunofluorescence

Raymond J Lim; Camelia Dumitras; Kostyantyn Krysan; Linh M Tran; Samantha Man; Steven M Dubinett; Bin Liu

doi:10.1016/j.xpro.2026.104570

Protocol for mapping tumor infiltration by murine T cells using multiplex immunofluorescence

STAR Protoc. 2026 Jun 19;7(2):104570. doi: 10.1016/j.xpro.2026.104570. Epub 2026 May 14.

Authors

Raymond J Lim¹, Camelia Dumitras², Kostyantyn Krysan³, Linh M Tran³, Samantha Man⁴, Steven M Dubinett⁵, Bin Liu⁶

Affiliations

¹ Division of Pulmonary and Critical Care, Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: raymondjohnlim@g.ucla.edu.
² Division of Pulmonary and Critical Care, Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: cdumitras@mednet.ucla.edu.
³ Division of Pulmonary and Critical Care, Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Medicine, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA.
⁴ Division of Pulmonary and Critical Care, Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁵ Division of Pulmonary and Critical Care, Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Medicine, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA; Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁶ Division of Pulmonary and Critical Care, Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: bliu@mednet.ucla.edu.

Abstract

Here, we present a 7-plex immunofluorescence staining protocol that enables the spatial analysis of murine tumor tissues to investigate the complex interplay between immune cells and the tumor microenvironment. We used a sequential antibody labeling strategy that identifies CD4⁺ and CD8⁺ T cells and characterizes their activation and regulatory states. This protocol also maps T cell infiltration within tumors, allowing researchers to investigate immune cell distribution, interactions, and the dynamic biology of the tumor immune landscape in murine tumor models. For complete details on the use and execution of this protocol, please refer to Lim et al.¹.

Keywords: Cancer; Cell Biology; Immunology.

MeSH terms

Animals
CD4-Positive T-Lymphocytes* / immunology
CD8-Positive T-Lymphocytes / immunology
Fluorescent Antibody Technique* / methods
Lymphocytes, Tumor-Infiltrating* / immunology
Mice
T-Lymphocytes* / immunology
Tumor Microenvironment / immunology